T cell activation induces a metabolic switch generating energy for proliferation, survival, and functions. We used non-invasive label-free two-photon fluorescence lifetime microscopy (2P-FLIM) to map the spatial and temporal dynamics of the metabolic NAD(P)H co-enzyme during T lymphocyte activation. This provides a readout of the OXPHOS and glycolysis rates at a single cell level. Analyzes were performed in the CD4+ leukemic T cell line Jurkat, and in human CD4+ primary T cells. Cells were activated on glass surfaces coated with activating antibodies mimicking immune synapse formation. Comparing the fraction of bound NAD(P)H between resting and activated T cells, we show that T cell activation induces a rapid switch toward glycolysis. This occurs after 10 minutes and remains stable for one hour. Three-dimensional analyzes revealed that the intracellular distribution of fraction of bound NAD(P)H increases at the immune synapse in activated cells. Finally, we show that fraction of bound NAD(P)H tends to negatively correlate with spreading of activated T cells, suggesting a link between actin remodeling and metabolic changes. This study highlights that 2P-FLIM measurement of fraction of bound NAD(P)H is well suited to follow a fast metabolic switch in 3D, in single T lymphocytes with subcellular resolution.